title: Comparative analysis of inactivated-state block of N-type (Ca(v)2.2) calcium channels.
abstract: OBJECTIVE
  The aim of this study was to compare a diverse set of peptide and small-molecule calcium channel blockers for inactivated-state block of native and recombinant N-type calcium channels using fluorescence-based and automated patch-clamp electrophysiology assays.
  
  
  METHODS
  The pharmacology of calcium channel blockers was determined at N-type channels in IMR-32 cells and in HEK cells overexpressing the inward rectifying K(+) channel Kir2.1. N-type channels were opened by increasing extracellular KCl. In the Kir2.1/N-type cell line the membrane potential could be modulated by adjusting the extracellular KCl, allowing determination of resting and inactivated-state block of N-type calcium channels. The potency and degree of state-dependent inhibition of these blockers were also determined by automated patch-clamp electrophysiology.
  
  
  RESULTS
  N-type-mediated calcium influx in IMR-32 cells was determined for a panel of blockers with IC(50) values of 0.001-7 μM and this positively correlated with inactivated-state block of recombinant channels measured using electrophysiology. The potency of several compounds was markedly weaker in the state-dependent fluorescence-based assay compared to the electrophysiology assay, although the degree of state-dependent blockade was comparable.
  
  
  CONCLUSIONS
  The present data demonstrate that fluorescence-based assays are suitable for assessing the ability of blockers to selectively interact with the inactivated state of the N-type channel.
authors.affiliation: Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Dept R4PM, Bldg. AP9A, 100 Abbott Park Road, Abbott Park, IL 60064-6125, USA. timothy.vortherms@abbott.com
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Vortherms
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Swensen
authors.forename: Wende
authors.initials: W
authors.lastname: Niforatos
authors.forename: James T
authors.initials: JT
authors.lastname: Limberis
authors.forename: Torben R
authors.initials: TR
authors.lastname: Neelands
authors.forename: Richard S
authors.initials: RS
authors.lastname: Janis
authors.forename: Rama
authors.initials: R
authors.lastname: Thimmapaya
authors.forename: Diana L
authors.initials: DL
authors.lastname: Donnelly-Roberts
authors.forename: Marian T
authors.initials: MT
authors.lastname: Namovic
authors.forename: Di
authors.initials: D
authors.lastname: Zhang
authors.forename: C
authors.initials: C
authors.lastname: Brent Putman
authors.forename: Ruth L
authors.initials: RL
authors.lastname: Martin
authors.forename: Carol S
authors.initials: CS
authors.lastname: Surowy
authors.forename: Michael F
authors.initials: MF
authors.lastname: Jarvis
authors.forename: Victoria E
authors.initials: VE
authors.lastname: Scott
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000378:metabolism*; D020864:Calcium Channels, N-Type / Q000378:metabolism* / Q000494:pharmacology; D002460:Cell Line; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D018408:Patch-Clamp Techniques
chemical_list: D002121:Calcium Channel Blockers; D020864:Calcium Channels, N-Type; D002118:Calcium